Cardiac glycosides are among the most widely used cardiotonic agents, despite a high incidence of toxicity. Their mechanism of action is thought to be exclusively an inhibition of the Na,K-ATPase. Other compelling evidence, however, suggests additional cellular actions, including direct actions on the sarcolemma and sarcoplasmic reticulum (SR), that contribute to their positive inotropic and/or toxic actions. The purpose of this project is to investigate new subcellular actions of several cardiotonic steroids on regulation of ion channels and contraction in heart. The following are the specific aims of this renewal application: l) to investigate how SRCRC activity is affected by a number of cardiotonic steroids and how the different structural components of these agents (including size and saturation of the lactone ring, presence of the carbohydrate moiety) might contribute to their positive inotropic actions; 2) to measure the effects of cardiotonic steroids to alter SRCRC activity in normal and failing human heart in order to determine if such an action might in fact contribute to its therapeutic or toxic actions in viva; 3) to determine if intracellular application of different cardiotonic steroids causes an increase in SR Ca2+ release, a positive inotropic effect and the development of toxicity; and 4) to study the effects of cardiotonic steroids on action potential configuration and ionic currents in order to determine if there is a direct action on specific sarcolemmal ion channels to contributes to their positive inotropic and/or toxic effects. Two complementary experimental approaches will be used. Single SRCRC activity will be measured by incorporation of SRCRC protein, isolated from cat ventricle, into artificial planar lipid bilayers. In addition, Ca2+i transients (measured with indo-1 fluorescence), ionic currents and contraction will be measured in isolated cat ventricular myocytes. The experiments will test the following Overall Hypothesis: Cardiac glycosides produce their cardiotonic actions via several mechanisms that include an intracellular action on SR Ca2+ release and direct effects on transmembrane ionic currents; these effects are independent from, but work in conjunction with, their known action to inhibit the sarcolemmal Na,K-ATPase. The results of these experiments will help to define the cellular mechanisms for the positive inotropic and toxic actions of cardiac glycosides. The significance of defining and distinguishing between the different cardiac glycoside effects on Ca2+i regulation via SRCRC activity and sarcolemmal ion channels is that it may then be possible to develop new agents that act selectively at these sites to retain their effectiveness in treating cardiac disease but whose toxic secondary actions on cardiac function might be curtailed or abolished.